PROJECT SUMMARY/ABSTRACT The long-term goal of this project is to determine which individuals are at greatest risk of developing clinically meaningful alloantibodies to platelet transfusion, and why, to enable targeted interventions to reduce these risks. Alloimmunization targeting donor MHC antigens is a common consequence of platelet transfusion and can cause serious harm including rejection of future transfusions or transplants. Measurements of the incidence of anti-MHC antibodies in platelet recipients vary widely, ranging from 7-55%. Many factors can influence alloimmunization outcomes, but one less studied area is the influence of the recipient's underlying health. The majority of the illnesses and medical interventions that necessitate transfusion have a profound impact on the immunological environment in which transfused donor antigens are encountered. Efforts to evaluate the role of patient health on alloimmunization have been limited as different groups of patients are treated with varying types and amounts of blood products. Here we propose to determine how different forms of immune modulation, common among transfusion recipients, influence the alloresponse to foreign MHC. An established murine model of transfusion-induced alloimmunization to MHC will be used to isolate the role of recipient health under controlled and standardized conditions. The central hypothesis is that the immunological environment established by the health of the recipient has a strong impact on both the magnitude and quality of the anti-MHC antibody response to allogeneic transfusion, driven by differences in B cell differentiation and T cell help. The specific aims are to evaluate the impact of inflammation or immunosuppressive therapies at the time of allogeneic transfusion on 1) alloantibody responses to MHC antigens, activation of allospecific B cells and the development of durable immunity; 2) the immunological environment and the quality of T cell help; and 3) the clinical significance and functional capabilities of alloantibodies generated under these different inflammatory or suppressive conditions. Three model interventions are included: chemotherapy (cancer treatment), LPS (bacterial infection), and poly(I:C) (viral infection). Antibodies against class I and class II MHC will be measured by isotype over time using our established assays. Through the use of MHC-tetramers, rare endogenous MHC-specific B cell populations will be examined in wild-type (non-transgenic/knock-out) mice under defined physiological conditions. Impact on cytokine milieu, T cell differentiation, and lymphocyte homeostasis will be determined. The clinical significance of alloantibodies will be assessed by their ability to drive rejection in models of platelet refractoriness and bone marrow transplantation. This work will identify classes of transfusion recipients at greatest risk for development of clinically meaningful anti-MHC antibodies, and the mechanisms driving these responses, which will inform future transfusion practice and development of new therapeutics.